VECTORS DIRECTING PERSISTENT MYELOID GENE EXPRESSION

指导持续性骨髓基因表达的载体

• 批准号: 6402749
• 负责人: John Peter O'Rourke
• 金额: $ 4.02万
• 依托单位: RESEARCH INST NATIONWIDE CHILDREN'S HOSP
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6402749
• 关键词: CD34 molecule; Retroviridae; SCID mouse; binding sites; cell differentiation; cell type; chromatin; embryo /fetus cell /tissue; flow cytometry; gene expression; gene induction /repression; gene therapy; genetic promoter element; green fluorescent proteins; hematopoietic stem cells; myeloid stem cell; northern blottings; nucleic acid repetitive sequence; polymerase chain reaction; technology /technique development; tissue /cell culture; transcription factor; transfection /expression vector; western blottings

Improvements have been made in retroviral mediated transduction of fetal tissues. However, in utero gene therapy, like other gene therapy applications, has been hampered by severely limited levels of transgene expression and a lack of persistent expression in differentiated cells due to promoter silencing mechanisms. Although some genetic therapy applications for genetic diseases of the hematopoietic system may not require cell specific transgene expression, for others targeted expression may be an essential prerequisite for treatment of these disorders. The long-term goal of the proposed research is the development novel retroyiral vectors which direct high level, persistent, and cell specific transgene expression in differentiated graniiloc and monocytes for use in our ongoing in utero gene therapy studies. We propose to investigate promoter regions from myeloid specific genes C/EBPepsilon and CD 11b to direct cell type specific transgene expression. To overcome the problem of low and transient transgene expression levels we propose to augment our cell specific expression vectors with tandem repeats of a myeloid enhancer region which may enhance promoter activity and/or chromatin insulators to eliminate chromatin silencin effects. These expression vectors will be investigated for their ability to direct high, protracted, myeloid specific transgene expression by transduction into human myeloid cell lines and primary human CD34+ cells followed by in vitro differentiation. Transplantation of transduced human CD 34+ cells into irradiated SCID mice to recapitulate human hematopoiesis and subsequent analysis of gene expression in differentiated hematopoietic cells will be performed. Finally, the vector will analyzed for their ability to direct high, protracted, myeloid specific transgene expression by injection of retroviral supernatant into fetal mice. The results of these studies will provide critical information regarding the feasibility of persistent, cell specific gene expression for use in the in utero treatment of inherited hematopoietic diseases.

在逆转录病毒介导的胎儿组织转导中已经取得了改进。然而，子宫内基因治疗，像其他基因治疗应用，已受到严重限制的水平的转基因表达和缺乏持久的表达在分化的细胞由于启动子沉默机制。尽管造血系统遗传疾病的一些遗传治疗应用可能不需要细胞特异性转基因表达，但对于其他遗传疾病，靶向表达可能是治疗这些疾病的必要先决条件。所提出的研究的长期目标是开发新的逆转录病毒载体，其在分化的粒细胞和单核细胞中指导高水平、持续和细胞特异性的转基因表达，用于我们正在进行的子宫内基因治疗研究。 我们建议研究髓系特异性基因C/EBPeptide和CD 11b的启动子区域，以指导细胞类型特异性转基因表达。为了克服低的和瞬时的转基因表达水平的问题，我们提出用髓样增强子区域的串联重复序列来增强我们的细胞特异性表达载体，所述髓样增强子区域可以增强启动子活性和/或染色质绝缘子以消除染色质沉默作用。将研究这些表达载体通过转导至人骨髓细胞系和原代人CD 34+细胞中，随后进行体外分化，来指导高、持久、骨髓特异性转基因表达的能力。将转导的人CD 34+细胞移植到经辐照的SCID小鼠中以重现人造血，并随后分析分化造血细胞中的基因表达。 最后，通过将逆转录病毒上清液注射到胎鼠中，分析载体指导高、持久、骨髓特异性转基因表达的能力。这些研究的结果将提供关键信息的可行性，持续的，细胞特异性基因表达用于在子宫内治疗遗传性造血疾病。